Tuberculosis vaccine

ABSTRACT

A method of detecting an anti-mycobacterial CD8 T cell response comprising contacting a population of CD8 T cells of an individual with one or more peptides selected from the peptides represented by SEQ ID NO: 3, 4, 7, 8, 9, 10, 11 or 12, and, optionally, one or two further peptides represented by SEQ ID NO: 1 and/or 2, wherein one or more of said peptides may be substituted by an analogue which binds a T cell receptor which recognises the corresponding substituted peptide, and determining whether CD8 T cells of the CD8 T cell population recognize the peptide(s).  
     The invention also provides a method of vaccinating against infection by a mycobacterium, wherein the vaccination leads to a CD8 T cell response, comprising administering (i) a CD8 T cell epitope of a mycobacterium protein, (ii) an analogue of the epitope which is capable of inhibiting the binding of the epitope to a T cell receptor, (iii) a precursor or (i) or (ii) which is capable of being processed to provide (i) or (ii), or (iv) a polynucleotide which is capable of being expressed to provide (i), (ii) or (iii).

FIELD OF THE INVENTION

[0001] The invention relates to a method of detecting ananti-mycobacterial CD8 T cell response and a method of vaccinationagainst mycobacterial infection using peptides and proteins containingmycobacterial CD8 T cell epitopes. The mycobacterium may beM.tuberculosis.

BACKGROUND OF THE INVENTION

[0002] The current live attenuated vaccine for tuberculosis, BCG, hasvariable and limited efficacy in tuberculosis-endemic regions For avaccine to be effective it must cause the generation of a strong immuneresponse against M. tuberculosis. Until now researchers in this fieldhave concentrated on CD 4 T cell and antibody responses toM.tuberculosis. A correlation has not been shown in humans betweeninfection with M.tuberculosis and the generation of a CD8 T cellresponse against specific M.tuberculosis antigens

SUMMARY OF THE INVENTION

[0003] Using an assay which detects release of IFN-γ from T cells, theinventors have shown that CD8 T cells specific for tuberculosis antigensexist during tuberculosis infection and have identified particular C D8T cell epitopes from ESAT-6 which are recognised. Such epitopes may beused to detect anti-mycobacterial CD8 T cells The inventors have alsoshown the presence of CD8 T cells specific for tuberculosis antigens inhealthy contacts suggesting that such cells may be protective againsttuberculosis.

[0004] Accordingly the invention provides a method of detecting ananti-mycobacterial CD8 T cell response comprising contacting apopulation of CD8 T cells of an individual with or more peptidesselected from the peptides represented by SEQ ID NO: 3, 4, 7, 8, 9, 10,11 or 12, and, optionally, one or two further peptides represented bySEQ ID NO. 1 and/or 2, wherein one or more of said peptides may besubstituted by an analogue which binds a T cell receptor that recognisesthe corresponding substituted peptide, and determining whether CD8 Tcells of the CD8 T cell population recognize the peptide(s)

[0005] The invention also provides a method of vaccinating againstinfection by a mycobacterium, wherein the vaccination leads to a CD8 Tcell response, comprising administering (i) a CD8 T cell epitope of amycobacterium protein, (ii) an analogue of the epitope which is capableof inhibiting the binding of the epitope to a T cell receptor, (iii) aprecursor of (i) or (ii) which is capable of being processed to provide(i) or (ii), or (iv) a polynucleotide which is capable of beingexpressed to provide (i), (ii) or (iii).

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1(A) shows numbers of circulating peptide-specific effectorsfrom peripheral blood of NPH54.

[0007]FIG. 1(B) shows that peptide G specific IFN-γ secreting T cellsare CD8+

[0008]FIG. 1(C) shows peptide E specific IFN-γ release by an STCL.

[0009]FIG. 2(A) shows peptide G specific cytolytic activity of CTL line4-1 from NPH54

[0010]FIG. 2(B) shows peptide G specific cytolytic activity of a CTLline from NPH97

[0011]FIG. 2(C) shows lysis of HLA-B52 matched heterologous targets(Akiba BCL) expressing endogenously processed ESAT-6.

[0012]FIG. 3 shows lysis of autologous macrophage targets by CTL fromNPH144.

[0013]FIG. 4 shows lysis of HLA-A11.01 matched heterologous BCL by CTLfrom NPH144

[0014]FIG. 5 shows lysis of HLA-A68 02 matched heterologous BCL by CTLfrom NPH130.

[0015]FIG. 6 shows lysis of autologous BCL by CTL from NPH172.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The term “epitope” as used herein includes analogues of theepitope, unless the context requires otherwise It is understood that theterm “peptide” as used herein also includes the analogue of that peptide(which may not be a peptide as defined by the ordinary use of the term)unless the context requires otherwise.

[0017] The invention provides a method of detecting anti-mycobacterialCD8 T cell responses in an individual This may be done for the purposeof testing the efficacy of a vaccine or for the purpose of diagnosing amycobacterial infection. The invention also provides a method ofvaccination of an individual

[0018] The sequences of SEQ D NO's 1 to 12 are shown below

[0019] SEQ ID NO 1-AMASTEGNV

[0020] SEQ ID NO 2-LQNLARTI

[0021] SEQ ID NO.3-NVTSIHSLL

[0022] SEQ ID NO 4-ELNNALQNLART

[0023] SEQ ID NO 5-TATELNNALQNLART

[0024] SEQ ID NO 6-NLARTISEAGQAMAS

[0025] SEQ ID NO 7-SGSEAYQGVQQKWDA

[0026] SEQ ID NO:8-TATELNNAL

[0027] SEQ ID NO.9-RTISEAGQAM

[0028] SEQ ID NO: 10-AYQGVQQKW

[0029] SEQ ID NO 11-SEAYQGVQQ

[0030] SEQ ID NO 12 -SEAYQGVQQK

[0031] SEQ ID NO's 1 to 4 and 8 to 10 are the minimal epitopes which arerecognised by CD8 T cells SEQ ID NO's 8 to 10 represent minimal epitopeswhich are present in the peptides SEQ ID NO's 5 to 7 respectively Inaddition another CD8 T cell epitope is present in SEQ ID NO7 Theidentity of the additional epitope is still under investigation but islikely to be one the peptides represented by SEQ ID NO's 11 and 12

[0032] The Individual In Whom a CD8 T Cell Response is Detected or Whois Vaccinated

[0033] The individual is generally a mammal, such as a human or animal,typically one which can be naturally or artificially infected bymycobacterium. The individual may be a primate, cow, sheep, pig, badgeror rodent (e g a mouse or rat). The individual may test positive ornegative in a Manoux test. The individual may be at risk of amycobacterial infection, typically for socio-economic reasons or mayhave a genetic or acquired predisposition to mycobacterial infection.

[0034] In the method of detecting an anti-mycobacterial CD8 T cellresponse the individual has preferably been vaccinated with a vaccineintended to provided protection against a mycobacterial infection Thevaccine generally comprises proteins comprising mycobacterial sequence.The vaccine may comprise any of the polypeptides, polynucleotides orcompositions discussed herein The vaccination will generally have takenplace within a year before the method is carried out, for example withinsix months or within a month before the method is carried out.

[0035] The Epitope Used in the Detection or Vaccination Method

[0036] The epitope which is used in the detection method or vaccinationis of a mycobacterium protein. Such a mycobacterium is typicallypathogenic and capable of infecting mammals, such as those mammalsdiscussed above. The mycobacterium is typically M. tuberculosis, but mayalso be M. marinum, M. kansasii or M. bovis.

[0037] The mycobacterium protein containing the epitope is typically onewhich is secreted from the mycobacterium, and is preferably ESAT-6. Suchan FSAT-6 protein is generally homologous to ESAT6 of M.tuberculosis.

[0038] The epitope used may be selected from the minimal epitopesrepresented by or present in any one of SEQ ID NO's 1 to 12

[0039] The Detection Method The CD8 T cells which recognise the peptidein the detection method have generally been pre-sensitised in vivo toantigen from a vaccine or from a mycobacterium infection Theseantigen-experienced T cells are generally present in the peripheralblood of a host which has been exposed to the antigen at a frequency of1 to 10⁶ to 1 in 10³ peripheral blood mononuclear cells (PBMCs)

[0040] In the method the T cells can be contacted with the peptides invitro or in vivo, and determining whether the T cells recognise thepeptide can be done in vitro or in vivo.

[0041] Determination of whether the T cells recognise the peptide isgenerally done by detecting a change in the state of the T cells in thepresence of the peptide or determining whether the T cells bind thepeptide The change in state is generally caused by antigen specificfunctional activity of the T cell after the T cell receptor binds thepeptide Generally when binding the T cell receptor the peptide is boundto an MHC class I molecule, which may be present on the surface of aPBMC or an antigen presenting cell (APC) I

[0042] The change in state of the T cell may be the start of or increasein secretion of a substance from the T cell, such as a cytokine,especially IFN-γ, IL-2 or TNF-α Determination of IFN-γ secretion isparticularly preferred. The substance can typically be detected byallowing it to bind to a specific binding agent and then measuring thepresence of the specific binding agent/substance complex The specificbinding agent is typically an antibody, such as polyclonal or monoclonalantibodies Antibodies to cytokines are commercially available, or can bemade using standard techniques

[0043] Typically the specific binding agent is immobilised on a solidsupport After the substance is allowed to bind the solid support canoptionally be washed to remove material which is not specifically boundto the agent The agent/substance complex may be detected by using asecond binding agent which will bind the complex Typically the secondagent binds the substance at a site which is different from the sitewhich binds the first agent. The second agent is preferably an antibodyand is labelled directly or indirectly by a detectable label

[0044] Thus the second agent may be detected by a third agent which istypically labelled directly or indirectly by a detectable label Forexample the second agent may comprise a biotin moiety, allowingdetection by a third agent which comprises a streptavidin moiety andtypically alkaline phosphate as a detectable label.

[0045] In one embodiment the detection system which is used is theex-vivo ELISPOT assay described in WO 98123960. In that assay IFN-γsecreted from the T cell is bound by a first IFN-γ specific antibodywhich is immobilised on a solid support The bound IFN-γ is then detectedusing a second IFN-γ specific antibody which is labelled with adetectable label Such a labelled antibody can be obtained from MABTECH(Stockholm, Sweden). Other detectable labels which can be used arediscussed below The ELISPOT assay may be used with freshly isolated Tcells

[0046] The change in state of the T cell which can be measured may bethe increase in the uptake of substances by the T cell, such as theuptake of thymidine The change in state may be an increase in the sizeof the T cells, or proliferation of the T cells, or a change in cellsurface markers on the T cell.

[0047] Generally the T cells which are used in the method are taken fromthe host i a blood sample, although other types of samples which containT cells can be used. The sample may be added directly to the assay ormay be processed first. Typically the processing may comprise dilutingof the sample, for example with water or buffer. Typically the sample isdiluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold.

[0048] The processing may comprise separation of components of thesample Typically mononuclear cells (MCs) are separated from the samplesThe MCs will comprise the T cells and APCs Thus in the method the APCspresent in the separated MCs can present the peptide to the T cells. Inanother embodiment only T cells, such as only CD8 T cells, can bepurified from the sample. PBMCs, MCs and T cells can be separated fromthe sample using techniques known in the art, such as those described in(2).

[0049] In one embodiment the T cells used in the assays are in the formof unprocessed or diluted samples, or are preferably freshly isolated Tcells (such as in the form of freshly isolated MCs or PBMCs) which areused directly ex vivo, i e they are not cultured before being used inthe method However the T cells can be cultured before use, for examplein the presence of one or more of the peptides, and generally alsoexogenous growth promoting cytokines During culturing the peptides aretypically present on the surface of cells such as APCs Pre-culturing ofthe T cells may lead to an increase in the sensitivity of the method.Thus the T cells can be converted into cell lines, such as short termcell lines (for example as described in (4))

[0050] The APC used in the method may be any cell which has MHC class Imolecules on its surface It may or may not be a specialised antigenpresenting cell, such as a B cell, dendritic cell or macrophage The APCused in the method may from the same host as the T cell or from adifferent host The APC may be a naturally occurring APC or an artificialAPC The APC is capable of presenting the peptide to a T cell It istypically separated from the same sample as the T cell and is typicallyco-purified with the T cell. Thus the APC may be present in MCs or PBMCsThe APC is typically a freshly isolated ex vivo cell or a cultured cellIt may be in the form of a cell line, such as a short term orimmortalised cell line The APC may express empty MHC class I moleculeson its surface

[0051] Typically in the method the T cells derived from the sample canbe placed into an assay with all the peptides (i.e a pool of thepeptides) which it is intended to test (the relevant panel) or the Tcells can be divided and placed into separate assays each of whichcontain one or more of the peptides In the in vivo embodiment of thedetection method the relevant peptide(s) will of course be administeredto the host Typically one or more, or all, of the peptides representedby SEQ ID NO's 3, 4, 8, 9 and 10 are also used in the method In anotherembodiment only the peptides represented by SEQ ID) NO's 1, 2, 3, 4, 8,9, 10 and one of 11 or 12 are used in the method.

[0052] The invention also provides the peptides such as two or more ofany of the peptides mentioned herein (for example in any of thecombinations mentioned herein) for simultaneous, separate or sequentialuse

[0053] In one embodiment peptide per se is added directly to an assaycomprising T cells and APCs. As discussed above the T cells and APCs insuch an assay could be in the form of MCs When peptides which can berecognised by the T cell without the need for presentation by APCs areused then APCs are not required Analogues which mimic the originalpeptide bound to a MHC molecule are an example of such a peptide.

[0054] In one embodiment the peptide is provided to the presenting cellin the absence of the T cell This cell is then provided to the T cell,typically after being allowed to present the peptide on its surface. Thepeptide may have been taken up inside the cell and presented, or simplybe taken up onto the surface without entering inside the cell

[0055] The duration for which the peptide is contacted with the T cellswill vary depending on the method used for determining recognition ofthe peptide, Typically 10⁵ to 10⁷, preferably 5×10⁵ to 10⁶ PBMCs areadded to each assay In the case where peptide is added directly to theassay its concentration is typically from 10⁻¹ to 10³ μg/ml, preferably0.5 to 50 μg/ml or 1 to 10 μg/ml. ,

[0056] Typically the length of time for which the T cells are incubatedwith the peptide is from 4 to 24 hours, preferably 6 to 16 hours. Whenusing ex vivo PBMCs it has been found that 0.3×10⁶ PBMCs can beincubated in 10 μg/ml of peptide for 12 hours at 37° C.

[0057] The determination of the recognition of the peptide by the Tcells may be done by measuring the binding of the peptide to the T cellsTypically T cells which bind the peptide can be sorted based on thisbinding, for example using a FACS technique The detection of thepresence of T cells which recognise the peptide will be deemed to occurif the frequency of cells sorted using the peptide is above a ‘control’value The frequency of antigen-experienced T cells is generally 1 in 10⁶to 1 in 10³, and therefore whether or not the sorted cells areantigen-experienced T cells can be determined

[0058] The determination of the recognition of the peptide by the Tcells may be measured in vivo Typically the peptide is administered tothe individual and then a response which indicates recognition of thepeptide may be measured. In one embodiment the peptide is administeredintradermally, typically in a similar manner to the Mantoux test. Thepeptide may be administered epidermally The peptide is typicallyadministered by needle, such as by injection, but can be administered byother methods such as ballistics, for example the ballistics techniqueswhich have been used to deliver nucleic acids EP-A-0693 119 describestechniques which can typically be used to administer the peptideTypically from 0.001 to 1000 μg, for example from 0.01 to 100 μg or 0 1to 10 μg of peptide is administered.

[0059] Alternatively an agent can be administered which is capable ofproviding the peptides in vivo Thus a polynucleotide capable ofexpressing the peptide can be administered, typically in any of the waysdescribed above for the administration of the peptide. Thepolynucleotide typically has any of the characteristics of thepolynucleotide which is discussed below Peptide is expressed from thepolynucleotide in vivo and recognition of the peptide in vivo may bemeasured Typically from 0.001 to 1000 μg, for example from 0.01 to 100μg or 0.1 to 10 μg of polynucleotide is administered.

[0060] The Vaccination Method

[0061] As discussed above in the method of vaccination a CD8 T cellepitope, an analogue of the epitope, a precursor which may be processedto provide the epitope or analogue, or a polynucleotide which is capableof being expressed to provide the epitope, analogue or precursor isadministered

[0062] The precursor may comprise the sequence of or be the same as thesequence of a mycobacterium protein or a fragment of such a protein Sucha fragment is typically at least 8 amino acids long, for example 9, 10,11, 12 amino acids long, such as at least 20, 30, 50 or 80 amino acidslong The precursor may thus comprise sequence N-terminal and/orC-terminal to the sequence of the fragment The precursor may be anatural protein, a fragment thereof or a non-natural protein. ThusESAT-6 or a fragment of ESAT-6 may employed in the vaccination.

[0063] The precursor is either a peptide or non-peptide or may compriseboth peptide and non-peptide portions The precursor typically comprises1, 2, 3, 4 or more CD8 epitopes which may be the same or differentTypically 1, 2, 3 or more linkers are present in the peptide separatingthe epitopes The linkers may be 1, 2, 3, 4 or more amino acids in lengthThus 1, 2, 3, or more, or all of the epitopes may be contiguous witheach other or separated from each other The precursor is typically 10 to2000 amino acids in length, such as 50 to 1000, or 200 to 500 aminoacids

[0064] The epitope is generally 8, 9, 10, 11, 12 or more amino acids inlength and is capable of binding an HLA class I A, B or C molecule, orthe equivalents of these molecules in a non-human animal. The epitope isgenerally able to bind an HLA class I molecule present in the individualor population which is to be vaccinated. The epitope is typicallycapable of binding HLA-A2, HLA-A11 (e.g HLA-Al 1.01), HLA-A68 (e.g.MA-A68 02), HLA-B7, BLA-B8, HLA-B3S,BLA-B52 or HLA-B53 or the equivalentmolecules in a non-human animal.

[0065] The sequence of the epitope may be any of the sequencesrepresented by SEQ ID NO 1, 2, 3, 4, 8, 9, 10, 11 or 12 or the sequenceof an epitope present in SEQ ID NO 7 (other than the SEQ ID NO 10sequence).

[0066] Generally the precursor is capable of being processed by anantigen presenting cell (APC) leading to the presentation of theepitopes bound to an MHC class I molecule on the surface of the APC

[0067] Methods of obtaining T cell responses are known in the artGenerally a CD8 T cell response can be obtained by vaccinating using anappropriate dose, route of administration, adjuvant (e g those in (25)or (26)) or delivery system. In one embodiment the delivery system iscapable of providing the epitope or precursor to an antigen presentingcell, for example in an intracellular location which allows presentationof the epitope or of epitopes derived from the precursor

[0068] Typically the delivery system comprises the polynucleotidediscussed below, for example in the form of a recombinant virus or DNAparticles. In one embodiment the epitope, precursor or polynucleotideare provided to an APC (such as any of the types of APCs discussedherein) ex vivo and the APC is then administered to the individual

[0069] The adjuvant may cause the epitope, precursor or polynucleotideto adopt a particulate form The adjuvant is typically a virus orvirus-like particle (such as a yeast Ty particle, e.g. as in (25)), anacrylic based microbead, a saponin (e.g a 3,28-O-bisglycoside quillaicacid) or an emulsion (e.g. oil in water or water in oil) such as soybeanemulsion (e.g as in 26).

[0070] The polynucleotide which expresses (i), (ii) or (iii) istypically DNA or RNA, and is single or double stranded Thepolynucleotide generally comprises coding sequence that encodes (i),(ii) or (ii). The coding sequence is typically operably linked to acontrol sequence capable of providing for expression of thepolynucleotide Thus typically the polynucleotide comprises 5′ and 3′ tothe coding sequence sequences which aid expression, such as aidingtranscription and/or translation of the coding sequence

[0071] The polynucleotide may be capable of expressing (i), (ii) or(iii) in a prokaryotic and/or eukaryotic cell The polynucleotide istypically capable of expressing (i), (ii) or (iii) in a mammalian cell,such as in the cells of any of the mammals discussed above. Thepolynucleotide may be capable of expressing (i), (ii) or (iii) in thecellular vector discussed below

[0072] In one embodiment the polynucleotide is present in a virus orcellular vector, such as a virus which is capable of stimulating a CD8Tcell response, such as a vaccinta virus (e.g. MVA or NYVAC)

[0073] The vaccination may be based on the specific epitopes discussedabove which are represented by or contained in SEQ ID NO's 1 to l12.Thus the invention also provides a method of vaccination which leads toa CD8 T cell response, the T cells of which are specific for a CD8epitope which is represented by SEQ ID NO's 1, 2, 3, 4, 8, 9, 10) 11 or12, or which is present in the sequence represented by SEQ ID NO:7comprising administering (i) a CD8 epitope which is represented by SEQID NO's 1, 2, 3 or 4, or which is present in the sequences representedby SEQ ID NO's 5, 6 or 7, (μ) an analogue of the epitope which iscapable of inhibiting the binding of the epitope to a T cell receptor,(iii) a precursor of (i) or (ii) which is capable of being processed toprovide (i) or (ii) excluding ESAT-6 or fragments of ESAT-6, or (iv) apolynucleotide which is capable of being expressed to provide (i), (ii)or (iii). (iii) may be the peptides represented by SEQ ID NO 5, 6 or 7.

[0074] The invention also provides these particular epitopes, analogues,precursors and polynucleotides represented by (i), (ii), (iii) and (iv),which may be in a pharmaceutically composition in association with apharmaceutically acceptable carner or diluent

[0075] As discussed above particular adjuvants or delivery systems maybe used which stimulate a CD8 T cell response. Thus the invention alsoprovides a vaccine comprising an adjuvant which stimulates a CD8 T cellresponse and any of the epitopes, analogues, precursors orpolynucleotides discussed above. The invention also provides theseepitopes, analogues, precursors or polynucleotides in association with adelivery system which is capable of stimulating a CD8 T cell response

[0076] Analogues of the Peptides used in the Detection or VaccinationMethod

[0077] The analogue of a peptide can bind to a T cell receptor whichrecognises the original peptide. Therefore generally when the analogueis added to T cells in the presence of the original peptide, typicallyalso in the presence of a presenting cell, the analogue inhibits therecognition of the original peptide The binding of the analogue to thesaid T cell receptors can be tested by standard techniques. Such T cellreceptors can be isolated from T cells which have been shown torecognise the peptide (e.g using the method of the invention) Optionallysuch T cells may be sorted based on their ability to recognise theoriginal peptide, for example using a FACS technique. Demonstration ofthe binding of the analogue to the T cell receptors can then shown bydetermining whether the T cell receptors inhibit the binding of theanalogue to a substance that binds the analogue, e.g. an antibody to theanalogue Typically the analogue is bound in an MHC class I molecule insuch an inhibition of binding assay

[0078] Typically the analogue inhibits the binding of the peptide to a Tcell receptor. In this case the amount of peptide which can bind the Tcell receptor in the presence of the analogue is decreased. This isbecause the analogue is able to bind the T cell receptor and thereforecompetes with the peptide for binding to the T cell receptor.

[0079] T cells for use in the above binding experiments can be isolatedfrom patients with mycobacterial infection or from individual who havebeen administered with an anti-mycobacterial vaccine (such as any suchvaccine mentioned herein) Whole ESAT-6 is not encompassed by the term‘analogue’

[0080] Other binding characteristics of the analogue are also the sameas the peptide, and thus typically the analogue binds to the same MHCclass I molecule which the peptide binds The analogue typically binds toantibodies specific for the peptide, and thus inhibits binding of thepeptide to such an antibody

[0081] The analogue is typically a peptide It may have homology with theoriginal peptide The analogue is typically at least 70% homologous tothe original peptide, preferably at least 80 or 90% homologous thereto,over the entire length of the original peptide. Methods of measuringprotein homology are well known in the art and it will be understood bythose of skill in the art that in the present context, homology iscalculated on the basis of amino acid identity (sometimes referred to as“hard homology”) For example the UWGCG Package provides the BESTFITprogram which can be used to calculate homology (for example used on itsdefault settings) (1)

[0082] The analogue may differ (be derived) from the original peptide bysubstitution, insertion or deletion, for example by 1, 2, 3, 4 or moresubstitutions, deletions or insertions The substitutions are preferably‘conservative’ These are defined according to the following Table. Aminoacids in the same block in the second column and preferably in the sameline in the third column may be substituted for each other ALIPHATICNon-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D EK R AROMATIC H F W Y

[0083] The analogue typically has a length of 8, 9, 10, 11 or 12 aminoacids Typically the amino acids in the analogue at the equivalentpositions to amino acids in the original peptide which contribute tobinding the MHC molecule or are responsible for the recognition by the Tcell receptor, are the same or are conserved.

[0084] Typically the analogue peptide comprises one or moremodifications, which may be natural post-translation modifications orartificial modifications The modification may provide a chemical moiety(typically by substitution of a hydrogen, e.g of a C—H bond), such as anamino, acetyl, hydroxy or halogen (e g. fluorine) group or carbohydrategroup Typically the modification is present on the N or C terminus

[0085] The analogue may comprise one or more non-natural amino acids,for example amino acids with a side chain different from natural aminoacids Generally, the non-natural amino acid will have an N terminusand/or a C terminus. The non-natural amino acid may be an L-or D-aminoacid

[0086] The analogue typically has a shape, size, flexibility orelectronic configuration which is substantially similar to the originalpeptide It is typically a derivative of the original peptide

[0087] In one embodiment the analogue is or mimics the original peptidebound to a MHC class I molecule The analogue may be or may mimic theoriginal peptide bound to 2, 3, 4 or more MHC class I moleculesassociated or bound to each other These MHC molecules may be boundtogether using a biotin/streptavidin based system, in which typically 2,3 or 4 biotin labelled MHC molecules bind to a streptavidin moiety Thisanalogue typically inhibits the binding of the peptide/MHC Class Icomplex to a T cell receptor or antibody which is specific for thecomplex.

[0088] The analogue is typically an antibody or a fragment of anantibody, such as a Fab or (Fab)₂ fragment.

[0089] The analogue may be immobilised on a solid support, particularlyan analogue which mimics peptide bound to a MHC molecule.

[0090] The analogue is typically designed by computational means andthen synthesised using methods known in the art. Alternatively theanalogue can be selected from a library of compounds. The library may bea combinatorial library or a display library, such as a phage displaylibrary The library of compounds may be expressed in the display libraryin the form of being bound to a MHC class I molecule, such as the MHCmolecule which the original peptide binds. Analogues are generallyselected from the library based on their ability to mimic the bindingcharacteristics of the original peptides. Thus they may be selectedbased on ability to bind a T cell receptor or antibody which recognisesthe original peptide

[0091] Kits Provided by the Invention

[0092] The invention also provides a kit for carrying out the methodcomprising one or more of the peptides or analogues and optionally ameans to detect the recognition of the peptide by the T cell. Typicallythe means to detect recognition allows or aids detection based on thetechniques discussed above.

[0093] Thus the means may allow detection of a substance secreted by theT cells after recognition. The kit may thus additionally include aspecific binding agent for the substance, such as an antibody. The agentis typically specific for IFN-γ. The agent is typically immobilised on asolid support. This means that after binding the agent the substancewill remain in the vicinity of the T cell which secreted it Thus ‘spots’of substance/agent complex are formed on the support, each spotrepresenting a T cell which is secreting the substance. Quantifying thespots, and typically comparing against a control, allows determinationof recognition of the peptide.

[0094] The kit may also comprise a means to detect the substance/agentcomplex A detectable change may occur in the agent itself after bindingthe substance, such as a colour change. Alternatively a second agentdirectly or indirectly labelled for detection may be allowed to bind thesubstance/agent complex to allow the determination of the spots Asdiscussed above the second agent may be specific for the substance, butbinds a different site on the substance than the first agent

[0095] The immobilised support may be a plate with wells, such as amicrotitre plate. Each assay can therefore be carried out in a separatewell in the plate.

[0096] The kit may additionally comprise medium for the T cells,detection agents or washing buffers to be used in the detection steps.The kit may additionally comprise reagents suitable for the separationfrom the sample, such as the separation of PBMCs or T cells from thesample The kit may be designed to allow detection of the T cellsdirectly in the sample without requiring any separation of thecomponents of the sample.

[0097] The kit may comprise an instrument which allows administration ofthe peptide, such as intradermal or epidermal administration Typicallysuch an instrument comprises one or more needles The instrument mayallow ballistic delivery of the peptide. The peptide in the kit may bein the form of a pharmaceutical composition

[0098] The kit may also comprise controls, such as positive or negativecontrols. The positive control may allow the detection system to betested Thus the positive control typically mimics recognition of thepeptide in any of the above methods. Typically in the kits designed todetermine recognition in vitro the positive control is a cytokine. Inthe kit designed to detect in vivo recognition of the peptide thepositive control may be antigen to which most individuals shouldresponse.

[0099] The kit may also comprise a means to take a sample containing Tcells from the host, such as a blood sample The kit may comprise a meansto separate mononuclear cells or T cells from a sample from the host.

[0100] Peptide, Polynucleotides and Antibodies Provided by the Invention

[0101] The invention also provides a peptide whose sequence isrepresented by any one of SEQ ID NO's 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12or an analogue thereof The invention provides a diagnostic product orpanel comprising one or more of these peptides typically in thecombinations discussed above Such a product is typically a compositionsuch as a pharmaceutical composition.

[0102] The invention also provides a polynucleotide which is capable ofexpression to provide a peptide comprising the sequence of SEQ ID NO. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or an analogue thereof. Typicallythe polynucleotide is DNA or RNA, and is single or double stranded Thepolynucleotide therefore comprises sequence which encodes the sequenceof SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. To the 5′ and 3′of this coding sequence the polynucleotide of the invention has sequenceor codons which are different from the sequence or codons 5′ and 3′ tothese sequences in the ESAT-6 gene. Therefore the polynucleotide of theinvention does not comprise the sequence coding for the whole of ESAT-6or fragments of ESAT-6, other than sequence coding for fragmentsrepresented by SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12

[0103]5′ and/or 3′ to the sequence encoding the peptide thepolynucleotide has coding or non-coding sequence Sequence 5′ and/or 3′to the coding sequence may comprise sequences which aid expression, suchas transcription and/or translation, of the sequence encoding thepeptide The polynucleotide may be capable of expressing the peptide in aprokaryotic or eukaryotic cell. In one embodiment the polynucleotide iscapable of expressing the peptide in a mammalian cell, such as a human,primate or rodent cell.

[0104] The polynucleotide may be incorporated into a replicable vectorSuch a vector is able to replicate in a suitable cell. The vector may bean expression vector In such a vector the polynucleotide of theinvention is operably linked to a control sequence which is capable ofproviding for the expression of the polynucleotide. The vector maycontain a selectable marker, such as the ampicillin resistance gene

[0105] The polynucleotide, peptide or antibody (see below) of theinvention, or the agents used in the method (for example in thedetection of substances secreted from T cells) may carry a detectablelabel Detectable labels which allow detection of the secreted substanceby visual inspection, optionally with the aid of an optical magnifyingmeans, are preferred Such a system is typically based on an enzyme labelwhich causes colour change in a substrate, for example alkalinephosphate causing a colour change in a substrate Such substrates arecommercially available, e g from BioRad Other suitable labels includeother enzymes such as peroxidase, or protein labels, such as biotin, orradioisotopes, such a ³²P or ³⁵S The above labels may be detected usingknown techniques

[0106] Polynucleotides, peptides or antibodies (see below) of theinvention may be in substantially purified form. They may be insubstantially isolated form, in which case they will generally compriseat least 90%, such as at least 95, 97 or 99% of the polynucleotide,peptide or antibody in the preparation The substantially isolatedpeptides which are not peptides (as defined in the normal sense of theword) generally comprise at least 90%, such as at least 95, 97 or 99% ofthe dry mass of the preparation. The polynucleotide, peptide or antibodyare typically substantially free of other cellular components orsubstantially free other mycobacterial cellular components Thepolynucleotide or peptide may be used in such a substantially isolated,purified or free form in the method or be present in such forms in thekit

[0107] The peptide or polynucleotide may be in the form of apharmaceutical composition which comprises the peptide or polynucleotideand a pharmaceutically acceptable carrier or diluent Suitable carriersand diluents include isotonic saline solutions, for examplephosphate-buffered saline, Typically the composition is formulated forintradermal or epidermal administration or for application by ballistictechniques Thus the peptide or polynucleotide may be associated with acarrier particle for ballistic delivery

[0108] The peptide of the invention can be made using standard syntheticchemistry techniques, such as by use of an automated synthesizer Thepeptide is typically made from a longer polypeptide e.g a fusionprotein, which polypeptide typically comprises the sequence of thepeptide The peptide may be derived from the polypeptide by for examplehydrolysing the polypeptide, such as using a protease, or by physicallybreaking the polypeptide The polypeptide is typically ESAT-6, which mayhave been expressed recombinantly

[0109] The peptide can also be made in a process comprising expressionof a polynucleotide, such as by expression of the polynucleotide of theinvention. The expressed polypeptide may be further processed to producethe peptide of the invention. Thus the peptide may be made in a processcomprising cultivating a cell transformed or transfected with anexpression vector described above under conditions to provide forexpression of the peptide or a polypeptide from which the peptide can bemade. The polynucleotide of the invention can be made using standardtechniques, such as by using a synthesiser.

[0110] The invention also provides use of a peptide or analogue of theinvention to produce an antibody specific to the peptide. This antibodyor any of the antibodies mentioned herein nay be produced by raisingantibody in a host animal Such antibodies will be specific to thepeptide or to the substances mentioned above which bind antibodies Thepeptide or substances are referred to as the ‘immunogen’ below Methodsof producing monoclonal and polyclonal antibodies are well-known. Amethod for producing a polyclonal antibody comprises immunising asuitable host animal, for example an experimental animal, with theimmunogen and isolating immunoglobulins from the serum The animal maytherefore be inoculated with the immunogen, blood subsequently removedfrom the animal and the IgG fraction purified A method for producing amonoclonal antibody comprises immortalising cells which produce thedesired antibody Hybridoma cells may be produced by fusing spleen cellsfrom an inoculated experimental animal with tumour cells (Kohler andMilstein (1975) Nature 256, 495-497)

[0111] An immortalized cell producing the desired antibody may beselected by a conventional procedure The hybridomas may be grown inculture or injected intraperitoneally for formation of ascites fluid orinto the blood stream of an allogenic host or immunocompromised hostHuman antibody may be prepared by in vitro immunisation of humanlymphocytes, followed by transformation of the lymphocytes withEpstein-Barr virus

[0112] For the production of both monoclonal and polyclonal antibodies,the experimental animal is suitably a goat, rabbit, rat or mouse. Ifdesired, the immunogen may be administered as a conjugate in which theimmunogen is coupled, for example via a side chain of one of the aminoacid residues, to a suitable carrier The carrier molecule is typically aphysiologically acceptable carrier. The antibody obtained may beisolated and, if desired, purified

[0113] The Identification of CD8 T Cell Epitopes

[0114] CD8 T cell epitopes of a mycobacterium protein may be identifiedby

[0115] (i) providing to a population of cells comprising CD8 T cells (a)a peptide that comprises a mycobacterial protein sequence, or (b),ananalogue of (a) that comprises a mimic of the mycobacterial proteinsequence; under conditions suitable for the presentation of (a) or (b)to the CDs T cells, and

[0116] (ii) determining whether the CD8 T cells recognise (a) or (b) bydetecting the expression of a substance by the T cells, the expressionof the substance indicating that the T cells have recognised (a) or (b),and that (a) or (b) contain a CDs epitope or a mimic of a CD8 epitope

[0117] The minimal epitope which binds to the MHC molecule duringrecognition by the T cell may be identified, or the method may merelyshow that a particular peptide contains such an epitope.

[0118] In the method a peptide which comprises mycobacterial sequencecan be provided to the T cell Typically such a peptide consists of orcomprises a fragment (at least 8 amino acids long, such as 10, 15, 20,30 or more amino acids long) of a mycobacterial protein Thus the peptidemay be a natural protein, such as a mycobacterium protein, or a fragmentthereof The peptide may be a non-natural protein, such as a fusionprotein

[0119] In one embodiment pools of different peptides are provided to theT cells If a particular pool is recognised then the peptides in eachpool can be individually provided to the T cell to determine whichpeptide was recognised In order to find the actual epitope which the Tcell recognises (the minimal epitope) the smallest portion ofmycobacterium sequence which the T cell recognises must be determined.

[0120] The T cell may instead be provided with an analogue of thepeptide, The analogue comprises a region which mimics the mycobacterialsequence, and thus will mimic any CD8 epitope present in themycobacterial sequence Such a mimic of an epitope binds (and isrecognised) by a T cell receptor which binds (and recognises) theepitope. Thus the mimic is capable of inhibiting the binding of theepitope to a T cell receptor which binds the epitope In one embodimentthe analogue is processed by the APC and presents the mimic bound to aMHC molecule to the T cell.

[0121] The analogue or region which mimics the epitope may have any ofthe characteristics of the analogue of the epitope discussed above Theanalogue or region of the peptide may be related to the peptide in anyof the ways the analogue of the epitope is related to the epitope, suchas having homology with the peptide Typically the analogue or regionmimics the binding characteristics of the peptide, and therefore willpreferably inhibit the binding of an antibody to the peptide

[0122] Determination of whether a T cell recognises the peptide isgenerally done by any of the methods discussed above in regard to thedetection method provided by the invention.

[0123] Thus in the method of the invention the peptide which ispresented to the T cell may been obtained by providing a mycobacteriumprotein or a protein that comprises mycobacterial protein sequence to anantigen presenting cell and allowing it to be processed to produce thepeptide

[0124] The precursor may thus itself be a polypeptide which comprises amycobacterial sequence or an analogue of the peptide which comprises amimic of the mycobacterial sequence The precursor may be a fragment ofESAT-6 which is at least 8 amino acids in length.

[0125] In one embodiment the peptide or precursor is provided to the APCby providing to the APC a polynucleotide which is capable of expressingthe peptide or precursor The term ‘precursor’ includes such apolynucleotide The polynucleotide may have any of the properties of thepolynucleotide discussed above which is used in the vaccination ordetection method, and therefore it may be present in a virus or cellularvector.

[0126] The epitopes or peptides identified in the method described abovecan be used to vaccinate individuals in the same manner as any of theepitopes or peptides described above Analogues of the epitopes orpeptides may be produced and used in the same manner as the analoguesdescribed above. The epitopes and peptides may also be present in thesame form as any of the epitopes or peptides described above, such asbeing substantially isolated or purified, or be in a pharmaceuticalcomposition with a pharmaceutically acceptable carrier or diluent, or bein a vaccine, such as a vaccine comprising the adjuvants or deliverysystems discussed herein

[0127] Administration

[0128] Any of the peptides, analogues, precursors or polynucleotidesdiscussed above in any form or in association with any other agentdiscussed above is included in the termed ‘vaccination agent’ below. Atherapeutically effective amount of such a vaccination agent may begiven to a human patient in need thereof The condition of a patientsuffering from a mycobacterium infection can therefore be improved byadministration of such a vaccination agent The vaccination agent may beadministered prophylactically to an individual who does not have amycobacterium infection in order to prevent the individual becominginfected

[0129] Thus the invention provides the vaccination agent for use in amethod of treating the human or animal body by therapy In particular theinvention provides use of the vaccination agent to vaccinate againstinfection by a mycobacterium, where wherein the vaccination leads to aCD8 T cell response. The invention provides the use of the vaccinationagent in the manufacture of a medicament for vaccinating againstinfection by a mycobacterium, wherein the vaccination leads to a CD8 Tcell response

[0130] Thus the invention provides a method of vaccinating an individualcomprising administering the vaccination agent to the individual

[0131] The vaccination agent is typically administered by any standardtechnique used for administering vaccines, such as by injection. It canbe administered by ballistics techniques, for example the ballisticstechniques which have been used to deliver nucleic acids EP-A-0693 119describes ballistics techniques which can be used to administer thevaccination agent

[0132] The vaccination agent may be in the form of a pharmaceuticalcomposition which comprises the vaccination agent and a pharmaceuticallyacceptable carrier or diluent. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.Typically the composition is formulated for parenteral, intravenous,intramuscular, subcutaneous, transdermal or oral administration

[0133] The dose of vaccination may be determined according to variousparameters, especially according to the substance used, the age, weightand condition of the patient to be treated, the route of administration,and the required regimen A physician will be able to determine therequired route of administration and dosage for any particular patient Asuitable dose may however be from 0.001 to 1000 μg, for example from0.01 to 100 μg or 0.1 to 10 μg of the vaccination agent

[0134] In the case of vaccination agents which are polynucleotidestransfection agents may also be administered to enhance the uptake ofthe polynucleotides by cells Example of suitable transfection agentsinclude cationic agents (for example calcium phosphate and DEAE-dextran)and lipofectants (for example lipofectam™ and transfectam™)

[0135] When the vaccination agent is a polynucleotide a which is in theform of a viral vector the amount of virus administered is in the rangeof from 10⁴ to 10⁸ pfu, preferably from 10⁵ to 10⁷ pfu, more preferablyabout 10⁶ pfu for herpes viral vectors and from 10⁶ to 10¹⁰ pfu,preferably from 10⁷ to 10⁹ pfu, more preferably about 10⁸ pfu foradenoviral vectors When injected, typically 1-2 ml of virus in apharmaceutically acceptable suitable carrier or diluent is administered

[0136] The following Examples illustrate the invention

EXAMPLE 1

[0137] Determination of CD8 T Cell Epitopes

[0138] The sequences of ESAT-6 and antigens 85A, 85B, and 85C ofM.tuberculosis were scanned with allele-specific peptide motifs for theHLA class I types HLA-A2, HLA-B7, HLA-B8, HLA-B35, HLA-B52, and HLA-53The seven congruent sequences found in ESAT-6 are shown in Table 1sequences congruent with HLA-B7, HLA-B35, and HLA-B53 were predicted tobe present in ESAT-6. TABLE 1 Peptides from ESAT-6 HLA class I allelePeptide motif Peptide Sequence Position HLA-A2 -L/I/M----V/L/I AGIEAAASAI 10-18 B AIQGNVTSI 17-25 C LLDEGKQSL 29-36 D ELNNALQNL 64-72 EAMASTEGNV 82-90 HLA-B8 --K-K--L/I F EQKQSLTKL 31-39 HLA-B52 -Q----I/V GLQNLARTI  69-76

[0139] A total of 49 peptides congruent with the motifs were synthesizedon solid phase on a synthesizer (Zinsser, Frankfurt) usingfluorenylmethoxy-carbonyl chemistry. Purity was confirmed by HPLC Thesecandidates epitopes were then sorted into pools according to HLA class Ialleles and used to stimulate peripheral blood mononuclear cells(PBMCs)from subjects with the corresponding MA class I allele Invitro-expanded populations of peptide-specific T cells (STCL's made asdescribed below) were then detected with two different assays ofeffector function In a preliminary study using the standard ⁵¹Cr releaseCTL assay we detected low levels of lytic activity against some peptidepools, but responses from the few remaining cells were too weak todefine epitopes. We therefore adapted a more sensitive enzyme-linkedimmunospot (ELISPOT) technique for detecting single cell interferon γ(IFN-γ) secretion We calibrated this assay against the ⁵¹Cr release CTLassay and found it an order of magnitude more sensitive for detectinglow numbers of cultured human CD8 peptide-specific T cells The followingexperiments have been performed using the ELISPOT assay.

EXAMPLE 2

[0140] The Patients Used in the Study

[0141] 39 adult patients and contacts with suitable HLA types werestudied Most subjects originated from the Indian subcontinent, Africa,or northern Europe The number of patients with different clinical typesof disease, as well as the number of healthy contacts, are listed inTable 2. TABLE 2 Numbers of healthy contacts and patients with differenttypes of clinical disease Clinical phenotype Subjects, n Pulmonary(active) 8 Pulmonary (inactive) 6 Pleural 2 Lymphadenitis 4Osteoarticular 5 Miliary 1 Gastrointestinal 1 Epididymo-orchitis 1Healthy contacts 11 

[0142] Most subjects were tuberculin skin test positive All contactsexcept one who was not tested, had a positive Heaf test of grade 2 ormore. All patients had at least 5 mm of cutaneous induration in responseto intradermal injection with 1 tuberculin unit of purified proteinderivative, except four who were not tested and four who were negative(three with old inactive pulmonary tuberculosis and one with activemilliary disease) The distribution of each of the 6 HLA class I allelesfor which we tested peptide pools was as follows HLA-A2 was present in26 subjects, HLA-B8 in 7 subjects, HLA-B7 in 6 subjects, HLA-B35 in 2subjects, BLA-B52 in 2 subjects, and HA-1B53 in 2 subjects.

[0143] Each subject was tested against the peptide pool (or pools if thesubject had more than one suitable HLA class I allele) once only Repeatblood samples were obtained and further testing carried out only if thedonor had responded on the first occasion.

[0144] NPH54 was diagnosed with tuberculosis on the basis of clinicalsymptoms, acute erythema nodosum and mediastinal lymph-adenopathy.Tuberculin skin test showed 25 mm induration 48 hr after intradermalinjection of 1 tuberculin unit of purified protein derivative Inaddition, a very high antibody titer of >1/6,250 in response to the 16kDa antigen of M.tuberculosis, helped to confirm active infection (6).For NPH97, the tuberculin skin test was positive and culture of aproximal phalangeal bone biopsy of the affected hand onLowenstein-Jensen medium grew M tuberculosis

[0145] Tissue Typing

[0146] Subjects were HLA typed serologically NPH 54, NPH97, WB, Akiba,and PG were additionally typed by amplication refractory mutation system(ARMS)-PCR using sequence-specific oligonucleotide primers (7)

EXAMPLE 3

[0147] Techniques

[0148] ELISPOT Assay for IFNγ

[0149] Ninety-six-well polyvinylidene difluoride (PVDF)-backed plates(Millipore) precoated with the anti-IFN-γmAB 1-D1K at 15 μg/l (MACBTECH,Stockholm) were washed with RPMI medium 1640 and blocked with R10 for 1hr at room temperature Short-term cell lines (STCLs), CTL lines, orclones were washed two times in RPMI medium 1640, resuspended in R10, andispensed at known input cell numbers per well in duplicate wellsPeptide was added either directly to the supernatant at a finalconcentration of 2 μM (free peptide) or presented on a B cell line (BCL)repulsed with 10 μM peptide for 1 hr at 37° C., and then washed threetimes in R10 Plates were incubated for 12 hr at 37° C., in 5% CO₂/95%air After washing six times with phosphate buffered saline (PBS) /0 05%Tween 20 to remove cells, plates were incubated for 3 hr with the secondbiotinylated anti-IFN-γ mAB 7-B6-1-biotin at 1 μg/nl (MABTECH) A furtherwash, as above, was followed by incubation with a 1.1,000 dilution ofstreptavidin-alkaline phosphate conjugate for 2 hr. After another wash,chromogenic alkaline phosphate substrate (Bio-Rad) was added to thewells, and 30 min later plates were washed with tap water After drying,spot-forming cells (SFC) were counted under X20 magnification For exvivo ELISPOT assays, 500,000 freshly isolated uncultured PBMCs were usedper well Responses were considered significant if a minimum of 10 SFCswere present per well and, additionally, this number was at least twicethat in control wells.

[0150] Generation of CD8⁺ T Cell Lines

[0151] STCLs were generated as described (4) Briefly PBMCs separatedfrom whole blood were prepulsed with 30-50 μM peptide for 1 hr in a cellpellet and then diluted up to 1×1- ⁶ cells/ml in RPMI medium 1640supplemented with 10% pooled heat-inactivated human AB serum (R10) and25 ng/ml rhIL-7 R & D Systems), and seeded at 200 μl per well in aU-bottomed 96-well plate Lymphocult-T (Biotest, Dreiech, Germany) wasadded to 10% of each well at regular intervals STCLs were assayed at day14, Lines 3-9, 3-10, and 3-20 were generated from individual STCLs bytwo rounds of restimulation (on days 14 and 21) with autologouspeptide-pulsed irradiated BCL at a 1 3 feeder responder ratio Line 4-1was generated by pooling four G specific STCLs (originally establishedwith CD4-depleted PBMCs), followed by restimulation with peptide-pulsedautologous BCL

[0152] Generation of CD8⁺ T Cell Clones

[0153] Enumeration of IFN-γ SFCs after CD4 and CD9 depletions indicatedthat the frequency of CD8 G specific T cells in NPH54-derivedmonospecific lines 3-9 and 3-20 was 1 44 and 1 67 CD8 T cells,respectively This indicated that for T cells seeded at one cell perwell, 1 5-2 3 specific clones should be generated per 100 wells seededThis quantitative data guided our cloning procedure for each line, 240wells were seeded at one cell per well (following CD4 depletion) Cloningmix consisted of three-way mixed lymphocyte reaction. 10% Lymphocult-Tphytohemagglutinin at 1 μg/ml, and G pulsed autologous irradiated washedBCL, with a total of 100,000 feeders per well. Clones were screened inELISPOT assays and three G specific clones were subsequently recoveredfrom line 3-9 and two from line 3-20 Clones were maintained bypeptide-pulsed autologous irradiated BCL restimulation andsupplementation with 10% Lymphocult-T and rhIL-7 at 25 ng/ml

[0154]⁵¹Cr Release Cytotoxicity Assays.

[0155] Standard chromium release assays were performed as described (8)In brief BCLs were labelled with 100 m Ci⁵¹Cr (Amersham), washed in RPMImedium 1640, and then pulsed with peptide as above and plated out at5,000 cells per well CTL, R10, or 5% Triton X-100 were added Test wellwere in duplicate, other wells in quadruplicate Plate were incubated for5 hr at 37°, in 5% CO₂/95% air and harvested supernatant read onfiltermats in an LKB 1205 beta-plate scintillation counter (Wallac,Gaithersburg, Md.) Background ⁵¹Cr release was less than 20% Percentlysis was calculated from the formula 100×(E−m)/(T−M), where E is theexperimental release, M is the spontaneous release, and T is the maximalrelease

[0156] Specific Cell Depletions

[0157] CD4 and CD8 T cells were depleted by 30 min incubation withanti-CD4 or anti-CD8 mAbs conjugated to ferrous beads, DYNABEADS M-450,(Dynal, Oslo) in 500 μl of R10 on ice Following dilution of up to 5 mlIn R10,the conjugate-coated cells were removed by a magnetic field. CD8T cell depletions were highly effective and were not toxic, becausethere was no detectable loss of viability in the depleted population andresponses of antigen 85-specific CD4 T cell lines were unaffected bydepletion

[0158] Recombinant Vaccinia Virus Construction

[0159] Construction of the r^(VV) is described in (9) In brief, theamplification product of the ESAT-6 gene obtained by PCR using theplasmid template pAA249 (P. Andersen, Staatens Seruminstitut,Copenhagen-S, Denmark) was cloned into plasmid p1108-tPA to createp1108-tPA-ESAT-6. Homologous recombination into the thymidine kinaselocus of vaccinia strain WR with cationic lipid transfection wasfollowed by selection of r^(VV) using mycophenolic acid A secondnegative control r^(VV) was also constructed by using p1108 minus thecoding sequence Recombinants were verified by sequencing and expressionconfirmed by PCR and capture ELISA.

EXAMPLE 4

[0160] Identification of ESAT-6-Specific Effector T Cells Direct fromPeripheral Blood

[0161] Two CD8 epitopes in ESAT-6 were identified The T cells from donorNPH54, who had tuberculosis mediastinal lymphadenitis, recognisedpeptides corresponding to both of these epitopes Uncultured PBMCsisolated at the time of the diagnosis from NPH54, who has HLA-B52 andHLA-A2 01, secreted IFNγ in response to an ESAT-6 derived peptide poolfor these class I alleles in an ex vivo ELISPOT assay The mean number ofIFNγ SFCs enumerated from 5×10⁵PBMCs in duplicate wells was 19 for theESAT-6 peptides compared with 2 in the control wells with no peptide Asubsequent assay tested freshly isolated PBMCs against each of theindividual peptides within the responding pools, IFNγ SFCs were detectedin response to peptides E and G, whose sequences were congruent with theHLA-B52 and BLA-A2 01 peptide motifs, respectively The frequency of E(1.50,000) and G (1.23,000) specific IFNγ SFCs is of the same order ofmagnitude as SFCs for HLA-A2 01-restricted influenza matrix epitope M158-66 (1 14,000) (FIG. 1A) Unrestimulated PBMCs from a second donor,NPH97, with tuberculosis osteomyelitis, also recognised the G peptide.This patent also has EA-B52 and HLA-A2.01, and the magnitude of the Gspecific response was similar to the response to the HLA-A2-restrictedinfluenza matrix epitope Single cell IFNγ release by freshly isolated Tcells in these short 12-hr ex vivo assays, employing no stimulus otherthan cognate peptide, indicated that these cells are highly likely to becirculating activated effector T cell (2)

[0162] We have identified CD8 HLA class I-restricted T cells specificfor epitopes in the M.tuberculosis protein ESAT-6 in 4 of 39 infectedindividuals This is almost certainly an underestimate of the actualprevalence of M.tuberculosis specific CD8 CTL in infected individualsfor the following reasons. First, we limited the search to epitopesrestricted through six HLA class I alleles Second, of the very largenumber of antigens secreted by M.tuberculosis, we studies only two Andthird, M.tuberculosis specific CD8 CTL are more likely to be found indraining lymph nodes or at the site of infection rather than in theperipheral circulation. Nevertheless, the frequencies of circulatingESAT-6 specific effectors in the peripheral blood of NPH54 approximatesto that for an influenza virus epitope This relatively high frequency ofCTL effectors specific for a single bacterial epitope is comparable tothat found in malaria, a protozoal disease, where there is substantialindirect evidence for a protective role for Plasmodium faiciparumspecific CTL (8) ESAT-6 specific effector among uncultured PBMCs weredetectable in the ex vivo ELISPOT assay in NPH54 with tuberculouslymphadenitis and in NPH97 with tuberculous osteomyelitis For the commonHLA class I allele, HLA-A2, the number of subjects studied is sufficientto permit a preliminary comparison of the prevalence of respondersbetween the different clinical subgroups. For donors with HLA-A2,, Especific IFN-γ secretion in freshly isolated PBMCs of STCLs was observedin 1 of 7 healthy contacts and 2 of 3 patients with lymphadenitis, butin none of 12 patients with other more disseminated forms of disease(pulmonary, pleural, and gastrointestinal) This distribution ofresponses suggests that response to ESAT-6 may be associated with animmune response capable of containing M.tuberculosis

[0163] The ELISPOT assay for IFN-γ release may measure an effectorfunction of more protective relevance than the ⁵¹ Cr releasecytotoxicity assay IFN-γ, a potent activator of macrophages, isessential for resistance to M.tuberculosis infection in mice (10, 11),whereas recent studies in perforin gene and Fas receptor gene knockoutmice indicate that the lytic activity of CD8 T cells is not required tocontrol virulent M.tuberculosis infection (12) Moreover, humanshomozygous for a point mutation in the IFN-γ receptor 1 gene, in whomcell surface expression of this receptor is absent, are highlysusceptible to fatal disseminated mycobacterial infection (13), andhuman T cell derived IFN-γ has recently been reported to inhibit theintracellular growth of M tuberculosis (Zhang, M, Gong, J, Lin,Y,Boylen, C T & Barnes, P F American Association of Immunologists JointMeeting, Jun. 2-6, 1996, New Orleans, La.). CD8 CTL derived IFN-γ may beespecially important both for cells lacking MHC class U molecules, e.gin the lung (14) and for macrophages where mycobacteria can evaderecognition during chronic infection by sequestering their antigens awayfrom sensitized CD4 T cells (15). Morever, infection of murinemacrophage cell lines with live M tuberculosis has recently been shownto down-regulate MHC class II expression while simultaneously enhancingthe presentation of exogenous soluble antigen through the MHC class Iantigen processing pathway(16).

EXAMPLE 5

[0164] ESAT-6 Epitope-Specific T Cells Are CD8

[0165] G specific T cell lines were generated from NPH54 and NPH97PBMCs. Depletion experiments demonstrated that the G specific T cellsare CD8 (FIG. 1B) Enumeration of IFN-γ SFCs in a 12 hr ELISPOT assay forIFN-γ with cell line 3-20 from donor NPH54 was performed and is shownAfter depletion of CD4 or CD8 cells, 20,000 cells were added to each ofa pair of duplicate wells and peptide G was added to a finalconcentration of 2 μM. the mean number of SFCs is shown in FIG. 1B NoSFCs were observed in the absence of peptide. CD8 cell depletioncompletely abrogates the response Similar results were obtained withcell line 3-9, Similar depletion studies on E specific STCLs from donorWB, a healthy contact with HLA-A2.01, confirmed that these E specific Tcells are also CD8 (FIG. 1C) After magnetic depletion of CD4 or CD8cells, 20,000 cells were added to each of a pair of duplicate wells in a12 hr ELISPOT assay and peptide added at 2 μM to the supernatant Themean number of IFN-γ SFCs for each pair of wells is shown in FIG. 1CCD4-depleted E specific STCLs from donor NPH43, a patient withlymphadenitis (class I HLA haplotype HLA-A2 01, HLA-A29, HLA-B7, andHLA-B51), recognised peptide presented through HLA-A2.01 on E-repulsedHLA-A2.01 matched heterologous BCL Ninety eight IFNγ SFCs wereenumerated in response to the E-pulsed BCL, compared with 48 IFNγ SFCsfor the unpulsed control BCL, the high backgrounds are probably due toalloresponse (24) Responses to E were transient and often undetectablein PBMCs from subsequent blood samples drawn later in the course oftherapy

[0166] No CDS epitopes were identified among any of the 42 peptides fromantigens 85A, 85B, or 85C Despite stimulation in vitro with nonamerpeptides, the resultant STCLs were all CD4 Interestingly, certainpeptides elicited IFN-γ secretion by freshly isolated uncultured CD4cells in 12-hr ex vivo ELISPOT assays

EXAMPLE 6

[0167] ES12-Specific CD8 T Cells Are MHC Class I-Restricted andRecognize Endogenously Processed Antigen

[0168] The epitope G was further characterized with specific lines andclones T cell recognition of peptide had until now relied onpresentation of G on autologous cells by adding free peptide to theELISPOT assay supernatant To demonstrate HLA class I restriction, Gpeptide was presented to clones derived from NPH54 with G-prepulsed BCLmatched (from Akiba)or mismatched (from donor PG)at B52; onlyHLA-B52-matched G prepulsed BCL were recognised, by cells pooled fromclones 3-1, 3-15, and 3-98 The matched BCL from Akiba are homozygous forHA-A24 and HLA-B52. The mismatched BCL from PG are HLA-A2 01, HLA-A3,HLA-B7 and HLA-B5 1 Assays were performed in duplicate wells with 5,000T cells and 50,000 B cells per well. Only the pair of duplicate wellswith G-pulsed HLA-B52 matched targets are positive the spots were sonumerous that they appeared confluent G-specific lines raised fromNPH97's PBMCs were similarly confirmed to be ELA-B52 restricted (datanot shown)

[0169] To show that the G-specific CD8 T cell clones were capable ofrecognising endogenously processed antigen, autologous BCL, infectedwith vaccinia virus recombinant for ESAT-6 (r^(VV)-ESAT-6) or a controllacking the ESAT-6 sequence, were used to stimulate cytokine release,BCL were infected the night before with the respective recombinantviruses at a multiplicity of infection (m o i) of 7 plaque forming unitsper cell in serum free medium, after 90 min, cells were diluted up to 1million/ml in R10 and incubated overnight Infected BCL (100,000) werethen added to each well along with 5,000 cloned T cells. Only clones3-1,3-15, and3-98 incubated with the ESAT-6 recombinantvaccinia-infected BCL secreted IFN-γ in the ELISPOT assay 3-15 gave inexcess of 450 SFCs The results with the other two clones, 3-1 and 3-98,were so strongly positive that the spots were confluent.

EXAMPLE 7

[0170]M tuberculosis Antigen-Specific CD8⁺ T Cells Are Cytolytic.

[0171] By using a sensitive measurement of cytokine release, wecharacterised CD8 T cells specific for G We now returned to conventional⁵¹Cr release assays to test whether these cells were also capable oflytic activity This was confirmed over a broad range of effector targetratios for CDS T cell lines from both NPH54 and NPH97 using G-pulsedheterologous HLA-B52 matched BCL as targets (FIG. 2A and B).Peptide-specific lysis was detectable even when the peptide prepulseconcentration was titrated as low as 1 nM (data not shown) FIG. 2(A)shows G specific cytolytic activity of CTL line 4-1 from donor NPH54 ina 5 hr ⁵¹Cr release cytotoxicity assay. Peptide specific lysis titrateddownwards with diminishing effector to target cell ratio, andnonspecific lysis of unpulsed targets was less than 5% Target cells wereheterologous HLA-B52 matched BCL (homozygous typing line, Akiba HLA-A24HLA-B52), prepulsed with 10 μM G. FIG. 2(B) shows G specific cytolyticactivity of a CTL line from donor NPH97 in a 5 hr ⁵¹Cr releasecytotoxicity assay The CTL line was generated by restimulation of 14days STCLs cultured as described above with G pulsed, washed, irradiatedautologous BCL. Peptide specific lysis titrated downwards with each 3fold diminution in effector-to-target cell ratio, and nonspecific lysisof unpulsed targets was less than 5%. Target cells were heterologousBLA-B52 matched BCL (Akiba) prepulsed with 10 μM of G

[0172] Finally, to establish whether G-specific CTL could also killtargets expressing endogenously processed antigen, we demonstratedHLA-B52-restricted lysis of heterologous HLA-B52-matched targets (AkibaBCL) infected with r^(VV)-ESAT-6 (FIG. 2C) Targets were infected withr^(VV)-ESAT-6 and r^(VV) control and were labelled with ⁵¹Cr thefollowing day. CTL line 4-1 raised against G specifically lysed ther^(VV)-ESAT-6 infected targets, lysis of r^(VV) control infected targetswas below 10%

[0173] Conclusions

[0174] The observation that G specific T cell lines and clones recognisetarget cells infected with vaccinia virus recombinant for ESAT-6indicates that this antigen can be endogenously processed through theMHC class I antigen processing pathway, resulting in the presentation ofthe epitope G through HLA-B52 Because responses to the M tuberculosisspecific peptide ES12 were elicited from freshly isolated,unrestimulated lymphocytes in an ex vivo assay, CD8 T cells must havebeen primed through recognition of processed antigen in vivo. This studythus provides evidence that in humans an M tuberculosis antigen isnaturally processed in vivo through the MHC class I pathway leading tothe induction of MHC class I restricted effector T cells. Furthersupport comes from preliminary data showing that human macrophagesinfected with M.tuberculoss in vitro art recognised by G specificHLA-B52 restricted CTL that suppress mycobacterial growth (data notshown)

[0175] Murine models, including studies with β₂-microglobulin geneknockout mice (17), TAP-1 null mutant mice (16), and adoptive transferexperiments with HSP-65 immunized mice (18) show that CD8 CTL areessential for protection against M tuberculosis infection CD8 T cellsalso constitute crucial effector mechanism in the protective immunityconferred by DNA vaccination against tuberculosis (19,20) It hastherefore been important to establish whether MHC class I restricted CD8CTL play a role in M tuberculosis infection in humans, for, if so, theirinduction could guide the rational design of subunit vaccines. However,it has proved very difficult to identify these cells in humans (21, 22)Indeed, the recent identification of human CD1 restricted CD8 T cellsspecific for M tuberculosis non peptide antigens has led to thesuggestion that disruption of CD1-mediated antigen presentation mayaccount for the enhanced susceptibility of β₂-microglobulin geneknockout mice to M.tuberculosis infection (23) However, this studydemonstrates the presence of classical MHC class I restricted CD8 CTLspecific for an M.tuberculosis protein antigen in infected individualsThese cells circulate at a relatively high frequency in peripheral bloodand freshly isolated, unrestimulated cells rapidly display effectorfunction within 12 hr of antigen contact Induction of MHC class Irestricted peptide specific CD8 CTL with new generation CTL inducingvaccines is now feasible. The phenotype and specificity of the cellsidentified here not only endorses efforts to develop CTL inducingvaccines against tuberculosis but also supports the candidacy of ESAT-6as a component of such vaccines.

EXAMPLE 8

[0176] The Identification of Further Epitopes

[0177] The ELISPOT assay was also used to show that the minimal epitopeNVTSIHSLL is recognised by CD8+ CTL tines from 2 different individualsone a patient with sputum smear positive pulmonary TB (NPH130, FIG. 5)and the other, remarkably, a healthy household contact (NPH172, FIG. 6).CTL lines from both subjects demonstrate vigorous cytolytic activity.

[0178] NPH 144, a patient with miliary and meningeal tuberculosisrecognises two different epitopes. CD8+CTL, lines specific for the 15mer NLARTISEAGQAMAS are strongly cytolytic The minimal epitope,RTISEAGQAM, has been defined T cell lines have been generated againstthis epitope from M.tuberculosis-infected individuals and these lineshave been shown to be CD8 positive (by immunomagnetic depletion prior tointerferon-gamma ELISPOT assay) and cytolytic (in chromium releasecytotoxicity assays) The epitope is restricted through HLA-B5702, asevidenced by T cell recognition of HLA-B5702-matched, but notmismatched, peptide-pulsed B cell lines in chromium release andinterferon-gamma ELISPOT assays CD8 T cells specific for this epitopehave been detected at high frequencies directly from the peripheralblood of M.tuberculosis-infected individuals in ex vivo interferon-gammaELISPOT assays

[0179] A second CD8+ T cell epitope is also recognised by NPH144 Thisepitope is contained within the 15 mer TATELNNALQNLART and isrepresented by TATELNNAL has been defined T cell lines and clones havebeen generated against this 9mer peptide from M.tuberculosis-infectedindividuals and these lines have been shown to be CD8 positive (byimmunomagnetic depletion prior to interferon-gamma ELISPOT assay andFACS analysis) and cytolytic (in chromium release cytotoxicity assays).The epitope is restricted through HLA-B3503, as evidenced by T cellrecognition of HLA-B3503 -matched, but not mismatched, peptide-pulsed Bcell lines in chromium release and interferon-gamma ELISPOT assays. CD8T cells specific for this epitope have been detected at high frequenciesdirectly from the peripheral blood of M tuberculosis-infectedindividuals in ex vivo interferon-gamma ELISPOT assays and bypeptide-MHC tetramers

EXAMPLE 9

[0180] ESAT-6-Specific CD8+ Effector T Cells Circulate At HighFrequencies in Some M.tuberculosis-Infected Individuals

[0181] In an ex-vivo 9-hr ELISPOT assay with freshly isolated PBL fromNPH144 responses to peptide ES 15 (TATELNNALQNLART) and ES 13(NLARTISEAGQAMAS) are found within 9-hrs of antigen contact (FIGS. 3 and4) This ex-vivo response is abrogated by CD8 depletion with Dynabeads.The frequency of circulating T cells specific for ES15 in NPH 144 rangesfrom 1/500-1/3,000. The mild diminution in the response with CD4depletion indicated that peptide ES15 may also be a target of IFN-γsecreting CD4+T cells in NPH144, as it is in many other tuberculosispatients and contacts.

[0182] T Cells Specific for Peptide ES15 Were Found to Infiltrate thePus of a Tuberculosis Cold Abscess

[0183] After 15 months anti-tuberculous chemotherapy, NPH144 remainsclinically generally well except for a recurrent pus-filled cold abscessat the site of the left supraclavicular lymph nodes Aspirated pus fromthis site is AFB smear negative and culture negative The pus consists oflymphocytes and neutrophils, 90% of these infiltrating white cells areapoptosed. An ex vivo ELISPOT assay with the surviving cells from theaspirated pus shows the presence of ES15 peptide-specific IFN-γsecreting T cells in the pus

[0184] Identification of the Minimal Epitope Within the PeptideSGSEAYQGVQQKWDA

[0185] Within the 15 mer peptide SGSEAYQGVQQKWDA, the minimal epitope,AYQGVQQKW has been defined. T cell lines have been generated againstthis epitope from M. tuberculosis-infected individuals and these lineshave been shown to be CD8 positive (by immunomagnetic depletion prior tointerferon-gamma ELISPOT assay) and cytolytic (in chromium releasecytotoxicity assays) The epitope is restricted through HLA-A24, asevidenced by T cell recognition of HLA-A24-matched, but not mismatched,peptide-pulsed B cell lines in chromium release and interferon-gammaELISPOT assays CDS T cells specific for this epitope have been detectedat high frequencies directly from the peripheral blood of Mtuberculosis-infected individuals in ex vivo interferon-gamma ELISPOTassays. Additionally, a second CD8 epitope exists with the 15 merSGSEAYQGVQQKWDA This epitope is restricted through HLA-B44, as evidencedby T cell recognition of HLA-B44-matched, but not mismatched,peptide-pulsed B cell lines in interferon-gamma ELISPOT assays Theidentity of the precise minimal epitope is under investigation and, onthe basis of the HLA-B44 peptide motif, is very likely to be eitherSEAYQGVQQ or SEAYQGVQQK

[0186] Discussion

[0187] We have identified 8 CD8 T cell epitopes in ESAT-6 These epitopesare restricted through MA-852 (for LQNLARTI), A2 (for AMASTEGNV), A68 02(for NVTSIHSLL), B3503 (for TATELNNAL), B5702 (for RTISEAGQAM), A24 (forAYQGVQQKW) and B44 (for the second epitope present in SGSEAYQGVQQKWDA)ESAT-6, only 95 amino acids in length, is thus extraordinarily rich inCD8+CTL epitopes Some of these epitopes moreover overlap with certainhuman CD4+epitopes (e.g. peptide ES15).

[0188] The frequency of ESAT-6-specific CD8+T cells is is very high insome individuals and these cells are capable of rapid effector functionin short duration ELISPOT assays for IFN-γ

[0189] We have shown that ESAT6-specific CD8+CTL can affect an unusualdelayed suppression of the growth of M tuberculosis in vitro but therole of these CD8+CTL in humans in vivo is not known One of oursubjects, NPH130, a healthy household contact, has a high frequency (1/1000 PB ) of CDS+CTL specific for the HLA-A68 02 restricted epitopeNVTSIHSLL, as demonstrated by ex vivo ELISPOT (FIG. 5). The presence ofsuch a high frequency of M.tuberculosis specific CD8+CTLs in an exposedbut healthy individual with a clinically undetectable bacillary loadraises the possibility that these T cells may, in some individuals, beassociated with containment of M.tuberculosis in vivo

References 1. Devereux et al (1984) Nucleic Acids Research 12, 387-395.

[0190] 2. Lalvani, A, et al. (1997) J.Exp. Med 1486, 859-865.

[0191] 3 Herr et al (1996) J. Immunological Methods. 191, 131-141

[0192] 4. Ota, K, et al. (1990) Nature (London) 346,183-187.

[0193] 5 Kohler and Milstein (1975) Nature 256, 495-497

[0194] 6. Wilkinson, R. J , et al. (1998) Ph D thesis (London Univ.,London)

[0195] 7 Krausa, P M, et al. (1995) Tissue Antigens 45, 223-231.

[0196] 8 Hill, A. V. S, et al. (1992) Nature (London) 360, 434-439

[0197] 9 Mackett, M (1995) in DNA Cloning 4 A Practical Approach, eds.Glover & Hanes, (IRL Oxford), p43

[0198] 10 Cooper, A, M., et al. (1993) J. Exp. Med 178, 2243-2247

[0199] 11 Flynn, J L., et al. (1993) J. Exp Med 78,2249-2254,

[0200] 12 Laochumroonvorapong, P, et al. (1997) Infect. Immun 65,127-132

[0201] 13 Newport, M. J., et al. (1996) N. Eng. J. Med. 335,1941-1949

[0202] 14 Orme, I M, (1997) in Host Response to Intracellular Pathogens,ed Kaufmann, S E H (Landes, Austin, Tex.), pp 115 -130.

[0203] 15 Pancholi, P., et al (1993) Scence 260 984-986

[0204] 16. Mazzacaro, R. J, et al. (1996) Proc Natl. Acad. Sci USA 93,11786-11791.

[0205] 17 Flynn, J. L, et al. (1992) Proc. Natl. Acad Aci USA 89,12013-12017.

[0206] 18 Silva, C L, Silva, M F, Pietro, R. C L R & Lowrie, D. B.(1994) Immnunology 83, 341-346.

[0207] 19. Huygen, K, et al (1996) Nat Med 2, 893-898

[0208] 20 Tascon, R E, et al, (1996) Nat. Med 21 888-892

[0209] 21. Turner J & Dockrell, H M (1996) Immunology 87, 339-342.

[0210] 22 DeLibeo, G., et al, (1988) Eur. J. Immunol 18, 59-64

[0211] 23 Stenger, S. et al, (1997) Science 276, 1684-1687

[0212] 24. Roche, P, et al. (1994) Infect. Immun 42, 5319-5326

[0213] 25 Allsopp, C E , et al (1996) Eur. J. Immunol 26, 1951-9

[0214] 26. Hioe, C. E, et al(1996) Vaccine 14, 412-8.

1. A method of detecting an anti-mycobacterial CD8 T cell responsecomprising contacting a population of CD8 T cells of an individual withone or more peptides selected from the peptides represented by SEQ D NO:3, 4, 7, 8, 9, 10, 11 or 12, and, optionally, one or two furtherpeptides represented by SEQ ID NO 1 and/or 2, wherein one or more ofsaid peptides may be substituted by an analogue which binds a T cellreceptor that recognises the corresponding substituted peptide, anddetermining whether CD8 T cells of the CDS T cell population recognizethe peptide(s)
 2. A method according to claim 1 wherein a peptide panelis employed consisting of the peptides represented by SEQ ID NO's 3, 4,8, 9 and 10, wherein one or more of these peptides may be substituted bysaid corresponding analogue 3 A method according to claim 1 wherein apeptide panel is employed consisting of the peptides represented by SEQID NO's 1, 2, 3, 4, 8, 9 and 10, wherein one or more of these peptidesmay be substituted by said corresponding analogue 4 A method accordingto claim 1 wherein any analogue which is used is (i) at least 70%homologous, preferably at least 80% homologous, more preferably at least90% homologous, to the entire corresponding substituted peptide, and/or(ii) has one or more deletions at the N-terminus and/or C-terminus incomparison to the corresponding substituted peptide, and/or (iii) hasone or more conservative substitutions compared to the correspondingsubstituted peptide. 5 A method according claim 1 in which therecognition of the peptide(s) by the CD8 T cells is determined bymeasuring secretion of a cytokine from the CD8 T cells 6 A methodaccording to claim 5 in which IFN-γ secretion from the T cells ismeasured. 7 A method according to claim 6 in which IFN-γ secretion fromthe CD8 T cells is determined by allowing secreted IFN-γ to bind animmobilised antibody specific to the cytokine and then determining thepresence of antibody/cytokine complex 8 A method according to claim 1 inwhich the CD8 T cells are freshly isolated ex vivo cells from peripheralblood. 9 A method according to claim 1 in which CD8 T cells arepre-cultured in vitro with the peptide(s). 10 A method according toclaim 1 in which the mycobacterium is M. tuberculosis 11 A methodaccording to claim 1 wherein the population of CD8 T cells is from anindividual to whom an anti-mycobacterial vaccine has been administered.12 A method according to claim 1 which s carried out in vitro. 13 Amethod according to claim 1 comprising administering one or morepolynucleotides capable of expressing in human cells peptides and/oranalogues as defined in claim 1 14 A kit for carrying out a methodaccording to claim 1 comprising one or more peptides selected from thepeptides represented by SEQ ID NO 3, 4, 7, 8, 9, 10, 11 or 12, and,optionally, one or two further peptides represented by SEQ ID NO 1and/or 2, wherein one or more of said peptides may be substituted by ananalogue which binds a T cell receptor which recognises thecorresponding substituted peptide, and optionally a means to detectrecognition of the peptide(s) by CD8 T cells. 15 A kit according toclaim 14 consisting of the peptides represented by SEQ ID NO's 3, 4, 8,9 and 10, wherein one or more of these peptides may be substituted bysaid corresponding analogue 16 A kit according to claim 14 consisting ofthe peptides represented by SEQ ID NO's 1, 2, 3, 4, 8, 9 and 10, whereinone or more of these peptides may be substituted by said correspondinganalogue. 17 A kit according to claim 14 which includes an antibody toIFN-γ. 18 A kit according to claim 17 wherein said antibody isimmobilised on a solid support and which optionally also includes ameans to detect any antibody/IFN-γ complex 19 A kit for carrying out amethod according to claim 13 comprising one or more polynucleotidescapable of expressing in human cells one or more peptides selected fromthe peptides represented by SEQ ID NO. 3, 4, 7, 8, 9, 10, 11 or 12, and,optionally, one or two further peptides represented by SEQ ID NO 1and/or 2, wherein one or more of said peptides may be substituted by ananalogue which binds a T cell receptor which recognises thecorresponding substituted peptide 20 A peptide whose sequence isrepresented by any one of SEQ ID NO's 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;or an analogue which binds a T cell receptor which recognises any one ofSEQ ID NO's 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 21 A pharmaceuticalcomposition a peptide or analogue as defined in claim 20 22 A method ofvaccinating against infection by a mycobacterium, wherein thevaccination leads to a CD8 T cell response, comprising administering (i)a CD8 T cell epitope of a mycobacterium protein, (ii) an analogue of theepitope which is capable of inhibiting the binding of the epitope to a Tcell receptor, (iii) a precursor of (i) or (ii) which is capable ofbeing processed to provide (i) or (ii), or (iv) a polynucleotide whichis capable of being expressed to provide (i), (ii) or (iii)
 23. A methodaccording to claim 22 in which the mycobacterial protein is from Mtuberculosis. 24 A method according to claim 22 wherein ESAT-6 or afragment of ESAT-6 is employed 25 A method of vaccination which leads toa CD8 T cell response, the CD8 T cells of which are specific for a CD8 Tcell epitope which is represented by any one of SEQ ID NO's 1, 2, 3, 4,8, 9, 10, 11 or 12, or which epitope is present in the sequencerepresented by SEQ ID NO 7, said method comprising administering (i) aCD8 T cell epitope which is represented by any one of SEQ ID NO's 1, 2,3, 4, 8, 9, 10, 11 or 12, or which is present in the sequencerepresented by SEQ ID NO. 7, (ii) an analogue of the epitope which iscapable of inhibiting the binding of the epitope to a T cell receptor,(iii) a precursor of (i) or (ii) which is capable of being processed toprovide (i) or (ii) excluding ESAT-6 or fragments of ESAT-6, or (iv) apolynucleotide which is capable of being expressed to provide (i), (ii)or (iii). 26 A pharmaceutical composition comprising an epitope,analogue, precursor or polynucleotide as defined in claim 25 and apharmaceutically acceptable carrier or diluent. 27 A vaccine comprisingan adjuvant which stimulates a CD8 T cell response and (i), (ii), (iii)or (iv) as defined in claims 22, or a vaccine comprising (ii), (ii),(iii) or (iv) as defined in claim 22 associated with a delivery systemcapable of stimulating a CD8 T cell response